2015-08-02T22:57:46ZCHARACTERIZING THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 2 UL21 PROTEIN IN VIRAL NUCLEAR EGRESShttp://hdl.handle.net/1974/13406
Title: CHARACTERIZING THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 2 UL21 PROTEIN IN VIRAL NUCLEAR EGRESS
Authors: Nassiri, Arash
Abstract: The herpes simplex virus type 2 (HSV-2) is an important human pathogen that is the main cause of genital herpes infections and has a significant and wide-ranging impact on human health. HSV-2 infections are one of the most common sexually transmitted diseases and are highly prevalent worldwide, thus making understanding their infection mechanisms all the more relevant. The UL21 gene, which is conserved amongst members of the Alphaherpesvirinae subfamily, encodes a tegument protein that is essential for HSV-2 propagation. More specifically, UL21 plays a critical role in the primary envelopment of capsids during nuclear egress, however the precise mechanism by which it does this is unclear. To investigate the role of UL21 in HSV-2 nuclear egress, we focused on events upstream of primary envelopment. First, we examined the role of UL21 in disrupting the localization of nuclear lamins during infection. Nuclear rim localization of lamins A, C, B1, and B2 was indistinguishable between the wild-type (WT), UL21 null (∆UL21), or repaired (∆UL21R) HSV-2 strains, suggesting UL21 is not implicated in the disassembly of nuclear lamins at the nuclear membrane. Additionally, UL21 did not influence the localization of the nuclear egress complex (NEC), composed of UL31 and UL34, insofar as cellular and virally-encoded UL31 and UL34 localization patterns were indistinguishable between the UL21 null, and repaired or wild-type strains. Lastly, in an attempt to characterize interacting partners for UL21, we focused on proteins implicated in nuclear egress or proteins that may function to recruit UL21 to the nuclear membrane, where it may carry out its essential role(s). While no interactions were detected between UL21 and key mediators of primary envelopment including UL31, UL34, or Us3, UL21 localization at the nuclear rim was enhanced by the overexpression of the inner nuclear membrane protein, LAP2β, suggesting a potential interaction between the two proteins. However, more experiments are warranted before any conclusive remarks can be made regarding such interaction. Taken together, these data have shed light on HSV-2 nuclear egress and have eliminated possible theories regarding the role of UL21 in HSV-2 primary envelopment.
Description: Thesis (Master, Microbiology & Immunology) -- Queen's University, 2015-07-16 09:10:57.3852015-07-17T04:00:00ZIDENTIFICATION OF NOVEL GENES INVOLVED IN N-GLYCOSYLATION AND REGULATION OF ARCHAELLA ASSEMBLY AND EXPLORATION OF THE EFFECT OF N-GLYCOSYLATION ON ARCHAELLA ASSEMBLY IN METHANOCOCCUS MARIPALUDIShttp://hdl.handle.net/1974/13405
Title: IDENTIFICATION OF NOVEL GENES INVOLVED IN N-GLYCOSYLATION AND REGULATION OF ARCHAELLA ASSEMBLY AND EXPLORATION OF THE EFFECT OF N-GLYCOSYLATION ON ARCHAELLA ASSEMBLY IN METHANOCOCCUS MARIPALUDIS
Authors: Ding, YAN
Abstract: In this thesis, using Methanococcus maripaludis as a model organism, significant, novel advances have been made to the understanding of the archaeal N-glycosylation pathway as well as archaella regulation and assembly. The archaellum is the swimming organelle in the third domain of life, the Archaea. Genes involved in archaellation are mainly clustered in the fla operon. The structural proteins of archaella, termed archaellins, are usually glycoproteins modified with N-glycans. In M. maripaludis, a euryarchaeon, all three archaellins FlaB1, FlaB2 and FlaB3 are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose exclusively found in this species. Using FlaB2 as a reporter protein, I genetically identified three novel genes, aglU, aglV and aglW, which are involved in the synthesis of the archaellin N-glycan. AglU, a putative threonine transferase, is thought to be responsible for the transfer of the threonine onto the third sugar residue in the N-glycan. AlgV and AglW, annotated as a methyltransferase and a UDP-glucose 4-epimerase, respectively, are involved in the biosynthesis of the unique terminal sugar.
Since it has been shown that disruption of the N-glycosylation pathway caused defects in archaella assembly or function, I explored the specific requirement of N-glycosylation of archaellins on archaellation by eliminating the four N-glycosylation sites in FlaB2 in all possible combinations. As many as three N-glycosylation sites could be removed from FlaB2 without interfering with archaella assembly or function. However, archaella could not be assembled when all four N-glycosylation sites in FlaB2 are eliminated.
While there is evidence that archaella synthesis in M. maripaludis is not constitutive, no transcriptional regulators had been identified for the fla operon in any euryarchaeon. I genetically identified and characterized ArnE as the first euryarchaeotic transcriptional activator of the fla operon. Deletion of arnE resulted in the cessation of the transcription and translation of the reporter gene flaB2 and translation of FlaB2 was restored when the mutant was complemented with arnE in trans. Electrophoretic mobility shift assays confirmed the specific binding of purified ArnE to DNA fragments from the fla promoter.
Description: Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2015-07-10 16:50:37.7192015-07-17T04:00:00ZCharacterization of the interactions of baculovirus transcription and replication regulators by bimolecular fluorescence complementationhttp://hdl.handle.net/1974/12628
Title: Characterization of the interactions of baculovirus transcription and replication regulators by bimolecular fluorescence complementation
Authors: Breznik, Jessica Ann
Abstract: Baculovirus gene expression is temporally divided into early and late stages. Viral late expression factors (LEFs) are essential for the transition between the early and late transcriptional stages, as well as for late gene expression. A virus-encoded RNA polymerase (RNApol) transcribes late and very late genes. Four LEFs comprise the core viral RNApol sufficient for in vitro transcription: LEF-4, LEF-8, LEF-9, and P47. LEF-4 has triphosphatase, guanylyltransferase, and ATPase activities. LEF-8 and LEF-9 contain catalytic motifs that are conserved in the active sites of other multisubunit RNApols. These motifs are required for in vitro baculovirus RNApol transcription. P47 has no demonstrated enzymatic activity. How these four subunits associate in vivo within the viral RNApol complex and initiate transcription is undetermined, as is whether its transcription mechanism requires the host TATA binding protein (TBP). Viral DNA replication coincides with the transition between early and late transcription and it is closely associated with the regulation of late gene expression. Two LEFs essential for viral DNA synthesis, LEF-3 and P143, have also been implicated in the regulation of baculovirus RNApol transcription. Bimolecular fluorescence complementation (BiFC) assays were adapted to characterize the in vivo interactions of the viral RNApol subunits LEF-4, LEF-8, LEF-9, and P47, in the context of viral infection, as well as to investigate their potential interactions with viral DNA replication proteins LEF-3 and P143, and host Spodoptera frugiperda TBP. The proteins of interest were modified by N-terminal fusions of the Venus1 (aa1-158) or Venus2 (aa159-239) fragments of the Venus fluorescent protein. Recombinant plasmids and baculoviruses were constructed. Nuclear fluorescence was detected from all BiFC assays investigating the self-association and inter-subunit interactions of the viral RNApol. BiFC assays also suggested that the viral RNApol proteins interact with LEF-3, P143, and TBP. The potential effects of endogenous viral proteins and viral DNA on the detected protein-protein interactions are discussed. The results highlight extensive interactions between the core baculovirus RNApol subunits, possible roles of viral DNA replication proteins in the nuclear localization and transcription mechanism of the viral RNApol, as well as the involvement of host TBP in in vivo late stage transcription.
Description: Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-11-30 13:56:45.7262014-12-01T05:00:00ZGENETIC ANALYSIS AND POST TRANSLATIONAL MODIFICATIONS OF TYPE IV PILI IN METHANOCOCCUS MARIPALUDIShttp://hdl.handle.net/1974/12518
Title: GENETIC ANALYSIS AND POST TRANSLATIONAL MODIFICATIONS OF TYPE IV PILI IN METHANOCOCCUS MARIPALUDIS
Authors: Balachandran Nair, DIVYA
Abstract: Methanococcus maripaludis has two different surface appendages that are considered to be type IV pili-like structures: the well-studied archaella as well as type IV pili. Though prevalent among various archaea, studies on type IV pili in any archaeon are extremely limited. In this thesis, many novel essential components necessary for type IV pili formation in M. maripaludis have been identified by gene deletion analysis coupled with electron microscopy. Several of these essential components were found in a previously identified eleven gene locus (mmp0231-mmp0241) containing three previously identified pilin-like genes and a prepilin peptidase. The genes in this locus were shown in this work, by reverse transcriptase-polymerase chain reaction experiments, to be arranged in a single operon. The remaining unstudied genes, except for mmp0231, were deleted and, with the exception of mmp0235 and mmp0238, found to be essential for piliation. Outside this locus, the conserved assembly ATPase (mmp0040; epdL) and, unusually, two tandem versions of the conserved type IV pilus platform protein (mmp0038 and mmp0039; epdJ and epdK) were identified. All three were shown to be essential for piliation. Furthermore, the gene encoding the major pilin structural protein (mmp1685; epdE) was identified at a separate locus. In addition, six other pilin-like genes (mmp0528, mmp0600, mmp0601, mmp0709, mmp0903, mmp1283) scattered around the genome were targeted for deletion, with electron microscopy of the individual deletion strains revealing that normal piliation was only affected when mmp1283 (epdD) was missing, suggesting it was a fourth minor pilin. Lastly, studies were conducted to examine the order of the two posttranslational modifications of pilins and archaellins. While it was determined that signal peptide cleavage and N-linked glycosylation of archaellins could occur independently of each other, this was not the case for pilins. Detection of epitope-tagged pilins expressed in various mutant backgrounds unable to carry out one, the other, or both posttranslational modifications, strongly suggested that pilins cannot be N-glycosylated unless the signal peptide is first removed. These studies reveal that the type IV pili of M. maripaludis are more complex than those of other studied archaea and also raise questions about how the cell is able to distinguish between the two types of type IV-pilin-like proteins, archaellins and pilins.
Description: Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2014-09-25 23:28:47.1242014-09-29T04:00:00Z